The Transcription Factors WRKY41 and WRKY53 Mediate Early Flowering Induced by the Novel Plant Growth Regulator Guvermectin in Arabidopsis thaliana

Flowering is a crucial stage for plant reproductive success; therefore, the regulation of plant flowering has been widely researched. Although multiple well-defined endogenous and exogenous flowering regulators have been reported, new ones are constantly being discovered. Here, we confirm that a novel plant growth regulator guvermectin (GV) induces early flowering in Arabidopsis. Interestingly, our genetic experiments newly demonstrated that WRKY41 and its homolog WRKY53 were involved in GV-accelerated flowering as positive flowering regulators. Overexpression of WRKY41 or WRKY53 resulted in an early flowering phenotype compared to the wild type (WT). In contrast, the w41/w53 double mutants showed a delay in GV-accelerated flowering. Gene expression analysis showed that flowering regulatory genes SOC1 and LFY were upregulated in GV-treated WT, 35S:WRKY41, and 35S:WRKY53 plants, but both declined in w41/w53 mutants with or without GV treatment. Meanwhile, biochemical assays confirmed that SOC1 and LFY were both direct targets of WRKY41 and WRKY53. Furthermore, the early flowering phenotype of 35S:WRKY41 lines was abolished in the soc1 or lfy background. Together, our results suggest that GV plays a function in promoting flowering, which was co-mediated by WRKY41 and WRKY53 acting as new flowering regulators by directly activating the transcription of SOC1 and LFY in Arabidopsis.


Introduction
Flowering is a key agronomic trait that plays an essential role during plant growth and development; it is also a major signal for the developmental transition from vegetative to reproductive growth [1]. Studies have shown that flowering is affected by multiple environmental conditions and endogenous developmental cues [1][2][3][4][5][6]. Over recent years, the molecular mechanisms and genetics of flowering have been reviewed in detail, and the main flowering pathways, such as vernalization, photoperiod, autonomous, age, and gibberellin (GA) pathways, have been revealed to explain flowering signaling in the model plant Arabidopsis thaliana [2,7]. Although flowering is affected by a variety of factors, genetic studies have shown that these flowering pathways converged on some key flowering regulatory genes, including CONSTANS (CO), FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), FLOWERING LOCUS C (FLC), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), APETALA1 (AP1), and LEAFY (LFY), to regulate flowering in plants [8][9][10][11][12].
In Arabidopsis, SOC1 acts as a key floral integrator gene. It encodes a MADS-box transcription factor that responds to multiple flowering pathways [13,14]. In the vegetative phase, SOC1 expression is suppressed by FLC and SHORT VEGETATIVE PHASE (SVP), but it can be induced by FT and GA in the floral transition stage [15][16][17]. SOC1, in collaboration with AGL24, can also regulate the floral meristem identity gene LFY by directly binding to its promoter [18]. SOC1 was also shown that be upregulated by the WRKY genes in an unknown way, such as AtWRKY75 [4]. LFY is an important flowering-time gene and plays a crucial role in determining flowering time [19,20]. LFY expression is rapidly increased upon floral induction, and it can be regulated by other genes, such as AtWRKY71 [21]. Meanwhile, LFY has also been proven to directly regulate the expression of the downstream gene AP1 [22].
Previous studies have shown that WRKY transcription factors (TFs) are involved in various physiological processes and play important roles in plant growth and development [23,24]. WRKY TFs are a type of DNA-binding protein identified by the peptide WRKYGQK at the N-terminus and a zinc finger motif at the C-terminus [25]. More than 70 WRKY genes have been found in Arabidopsis thaliana, and extensive molecular biology studies have shown that they are involved in plant growth and development, and biotic and abiotic stress signal transduction [23,[26][27][28][29]. For instance, AtWRKY8, AtWRKY33, AtWRKY38, AtWRKY62, and AtWRKY76 are involved in the response to fungal pathogens and abiotic stress [30][31][32][33]. AtWRKY71 interacts with EXB1 to control shoot branching by regulating RAX genes [34]. Moreover, it has been confirmed that WRKY genes, such as AtWRKY6, AtWRKY71, GsWRKY20, and OsWRKY11, positively regulate plant flowering [21,35].
Plant growth regulators (PGRs) play important roles in plant growth, development, and stress resistance, and have been widely used in agricultural production [36][37][38][39][40][41]. PGRs can also directly or indirectly affect plant flowering by acting as repressors or activators [9,10,42,43]. For example, brassinosteroid (BR) promotes the expression of the floral repressor FLC and its homologs to result in delayed flowering [44]. Physiological evidence indicates that cytokinin plays a role in floral transition and promotes flowering by activating the transcription of TSF and SOC1 [5]. Gibberellin (GA) plays multiple functions in plant development and promotes flowering by upregulating the floral meristem identity gene LFY, which is necessary for flower formation [9].  demonstrated that GA regulates floral transition through DELLA interacting directly with SQUAMOSA PROMOTER BINDING-LIKE (SPL).
Guvermectin (GV) is a novel N9-glucoside cytokinin compound identified from Streptomyces caniferus NEAU6 and has been successfully registered as a novel natural PGR (Registration Code: PD20212929) in China [45,46]. Notably, although GV is a nucleoside analog like cytokinin, the cytokinin receptor triple mutant ahk2-2ahk3-3cre1-12 still responds to GV treatment, indicating that GV plays a novel mechanism different from that of cytokinin [47]. Recently, the significant biological activity of GV in regulating plant growth and development has been confirmed. GV was shown that plays a role in promoting maize growth in high-temperature environments [45]. Moreover, GV also promoted root and hypocotyl growth in Arabidopsis and seed germination, tillering, and early maturing in rice [47]. Interestingly, we found that GV also has an effect in inducing early flowering in plants. However, little was known about the mechanism of GV acts in regulating plant flowering. Our biochemical and genetic experiments further demonstrated that the WRKY genes WRKY41 and WRKY53 are induced by GV and they co-mediate GV-accelerated flowering by directly binding to the SOC1 and LFY promoters and activating transcription in Arabidopsis. Meanwhile, this study provides a molecular basis for the application of GV to plants.

The Plant Regulator GV Can Accelerate Flowering
In our previous study, the significant biological activity in regulating plant growth and development of PGR guvermectin (GV) has been identified [45,47]. Moreover, in our biological function assays, GV was shown to have an effect in promoting flowering in Arabidopsis. GV (50 mg L −1 ) [45] was sprayed on two-week-old, wild-type (WT) plants grown under long-day (LD) conditions (16 h light/8 h dark) and treated with 0 mg L −1 GV as a control. The same treatment was performed on seven-week-old Arabidopsis grown under short days (SD) conditions (8 h light/16 h dark). As shown in Figure 1, GV treatment, significantly accelerated flowering ( Figure 1A and Table S1) compared to the control which was treated with 0 mg L −1 GV, as measured by days to flowering (DTF) ( Figure 1B) and rosette leaves number (RLN) ( Figure 1C). Similarly, GV also significantly accelerated Arabidopsis flowering under SD conditions ( Figure S1A,B and Table S1). The cytokinin receptor triple mutant ahk2-2 ahk3-3 cre1-12 showed a response to GV [47], moreover, three cytokinin receptor mutants ahk2/3, ahk2/4, and ahk3/4 all showed a response to GV and flowering early after GV treatment compared to the control ( Figure S1C,D), suggesting GV is different from cytokinin in regulating flowering. Together, these results suggest that GV accelerates flowering with a new mechanism in Arabidopsis.

The Plant Regulator GV Can Accelerate Flowering
In our previous study, the significant biological activity in regulating plant growth and development of PGR guvermectin (GV) has been identified [45,47]. Moreover, in our biological function assays, GV was shown to have an effect in promoting flowering in Arabidopsis. GV (50 mg L −1 ) [45] was sprayed on two-week-old, wild-type (WT) plants grown under long-day (LD) conditions (16 h light/8 h dark) and treated with 0 mg L −1 GV as a control. The same treatment was performed on seven-week-old Arabidopsis grown under short days (SD) conditions (8 h light/16 h dark). As shown in Figure 1, GV treatment, significantly accelerated flowering ( Figure 1A and Table S1) compared to the control which was treated with 0 mg L −1 GV, as measured by days to flowering (DTF) ( Figure 1B) and rosette leaves number (RLN) ( Figure 1C). Similarly, GV also significantly accelerated Arabidopsis flowering under SD conditions ( Figure S1A,B and Table S1). The cytokinin receptor triple mutant ahk2-2 ahk3-3 cre1-12 showed a response to GV [47], moreover, three cytokinin receptor mutants ahk2/3, ahk2/4, and ahk3/4 all showed a response to GV and flowering early after GV treatment compared to the control ( Figure  S1C,D), suggesting GV is different from cytokinin in regulating flowering. Together, these results suggest that GV accelerates flowering with a new mechanism in Arabidopsis. and RLN (C), were grown under LD conditions. Two-week-old plants were sprayed with 50 mg L −1 GV and CK, and the DTF and RLN were assessed, respectively. 25D: DTF of wild type (WT) plants grown under LD conditions. CK, control (treated with 0 mg L −1 GV). GV, guvermectin treatment. Three biological replicates were counted with similar results. Values are expressed as means ± SD (n = 30). A significant difference analysis was the Student's t-test (***, p < 0.001). Bar = 1 cm.

WRKY41 Is Significantly Upregulated by GV
To investigate the mechanism by which GV accelerates flowering in plants, RNA sequencing (RNA-seq) was performed on Arabidopsis plants four days after GV treatment. The plants were treated with 0 mg/L GV as a control. Our analysis's results showed that 1358 genes were significantly induced to express by GV ( Figure S2A and Dataset S1), meanwhile, the results of multiple types of gene qPCR consistent with RNAseq verified the reliability of the data ( Figure S2B,C). TFs were selected for analysis because of their important roles in signaling pathways controlling plant growth [24,48]. The transcriptome data analysis showed that multiple classes of TFs were affected by GV treatment, interestingly, WRKY TFs showed the largest change in transcript abundance (log2FoldChange ≥ 1.5) ( Figure S3A and Table S2). Notably, WRKY TFs have aroused our attention because they were previously reported to be involved in plant flowering, such as AtWRKY71 [21]. To further confirm these results, the expression levels of selected WRKY genes (Table S2) were determined by quantitative PCR (qPCR) at 0, 1, 3, 5, and 7 d after GV treatment, respectively. The results showed that the selected and RLN (C), were grown under LD conditions. Two-week-old plants were sprayed with 50 mg L −1 GV and CK, and the DTF and RLN were assessed, respectively. 25D: DTF of wild type (WT) plants grown under LD conditions. CK, control (treated with 0 mg L −1 GV). GV, guvermectin treatment. Three biological replicates were counted with similar results. Values are expressed as means ± SD (n = 30). A significant difference analysis was the Student's t-test (***, p < 0.001). Bar = 1 cm.

WRKY41 Is Significantly Upregulated by GV
To investigate the mechanism by which GV accelerates flowering in plants, RNA sequencing (RNA-seq) was performed on Arabidopsis plants four days after GV treatment. The plants were treated with 0 mg/L GV as a control. Our analysis's results showed that 1358 genes were significantly induced to express by GV ( Figure S2A and Dataset S1), meanwhile, the results of multiple types of gene qPCR consistent with RNA-seq verified the reliability of the data ( Figure S2B,C). TFs were selected for analysis because of their important roles in signaling pathways controlling plant growth [24,48]. The transcriptome data analysis showed that multiple classes of TFs were affected by GV treatment, interestingly, WRKY TFs showed the largest change in transcript abundance (log 2 FoldChange ≥ 1.5) ( Figure S3A and Table S2). Notably, WRKY TFs have aroused our attention because they were previously reported to be involved in plant flowering, such as AtWRKY71 [21]. To further confirm these results, the expression levels of selected WRKY genes (Table S2) were determined by quantitative PCR (qPCR) at 0, 1, 3, 5, and 7 d after GV treatment, respectively. The results showed that the selected WRKY genes were induced at different levels by GV. WRKY41 was the most highly upregulated in response to GV (Figure 2A) of the WRKY genes studied here ( Figure S3B-K). WRKY41 is reportedly involved in plant growth and development and is expressed in floral organs [49], implying that WRKY41 may play a role in flowering development. Thus, the significant induction of WRKY41 by GV suggested the possibility that WRKY41 involves in the process of GV-accelerated flowering. WRKY genes were induced at different levels by GV. WRKY41 was the most highly upregulated in response to GV (Figure 2A) of the WRKY genes studied here ( Figure  S3B-K). WRKY41 is reportedly involved in plant growth and development and is expressed in floral organs [49], implying that WRKY41 may play a role in flowering development. Thus, the significant induction of WRKY41 by GV suggested the possibility that WRKY41 involves in the process of GV-accelerated flowering.

WRKY41 and Its Homolog WRKY53 Play Roles in GV-Accelerated Flowering
To determine the biological functions of WRKY41 in regulating flowering, we generated two overexpression of WRKY41 ( Table S1), suggesting that WRKY41 has a role in flowering regulation. Notably, WRKY61

WRKY41 and Its Homolog WRKY53 Play Roles in GV-Accelerated Flowering
To determine the biological functions of WRKY41 in regulating flowering, we gener-  Table S1), suggesting that WRKY41 has a role in flowering regulation. Notably, WRKY61 showed a significantly elevated upon GV induction ( Figure S3B), However, the overexpression of WRKY61 does not affect flowering ( Figure S5A,B) indicating that it might not be involved in the regulation of flowering. In addition, the flowering phenotypes were indistinguishable between wrky41 and WT plants ( Figure 2F-H), implying that other WRKY genes must be involved. Importantly, GV-induced early flowering was attenuated in the wrky41 mutants compared with that in WT plants ( Figure 2G,H), suggesting that WRKY41 contributes to the process of GV-accelerated flowering.
A phylogenetic tree of WRKY TFs indicated that WRKY53 is the closest homolog to WRKY41 ( Figure S6), consistent with the description of Wu et al. [50]. The RNAseq and qPCR data both showed that WRKY53 levels were altered after GV treatment (Table S1 and Figure Table S1), suggesting that WRKY53 plays a role in regulating flowering. However, no differences in flowering phenotypes of wrky53 and the WT plants ( Figure 3F-H and Table S1) implied that still other WRKY genes must be involved. Interestingly, similar to the results of WRKY41, the GV-induced early flowering phenotype was also weakened in wrky53 compared to that in WT plants ( Figure 3G,H). This suggested that WRKY41 and WRKY53 may be functionally redundant. We therefore further explored the potential redundant function of WRKY41 and WRKY53 in regulating flowering. A double knockout mutant was generated for WRKY41 and WRKY53 using CRISPR/Cas9-mediated genome editing technology. Two homozygous lines, w41/w53-1 and w41/w53-2, were confirmed by sequencing ( Figure 4A,B). Both double mutant lines flowered later than the WT plants ( Figure 4C-E and Table S1), indicating that WRKY41 and WRKY53 indeed have redundant functions in regulating flowering. Notably, the GV-induced early flowering phenotypes in WT were not present in the w41/w53 mutants ( Figure 4C-E), implying that WRKY41 and WRKY53 play important roles in the process of GV-accelerated flowering. Taken together, these results suggest that WRKY53 functions redundantly with WRKY41 and they co-mediate GV-accelerated flowering.

WRKY41 and WRKY53 Activate the Transcription of SOC1 and LFY
The biological activity of GV in accelerating flowering has previously been confirmed. However, it remains to be verified whether flowering regulatory genes are involved in the process of GV flowering induction. Our transcriptome data analysis showed that many flower-regulated genes are differentially expressed after GV treatment ( Figure S8A). During flowering, the major flowering regulatory genes GI, CO, FT, SOC1, AP1, LFY, FLC, and TFL1 are regulated [9,14,51]. We collected samples at 0, 1, 3, 5, and 7 d after 50 mg L −1 GV and control (0 mg L −1 GV) treatment. Our results showed the transcript levels of the floral regulatory genes SOC1, LFY, and AP1 were significantly upregulated (2~6 foldchange) compared to the control after GV treatment in Arabidopsis

WRKY41 and WRKY53 Activate the Transcription of SOC1 and LFY
The biological activity of GV in accelerating flowering has previously been confirmed. However, it remains to be verified whether flowering regulatory genes are involved in the process of GV flowering induction. Our transcriptome data analysis showed that many flower-regulated genes are differentially expressed after GV treatment ( Figure S8A). During flowering, the major flowering regulatory genes GI, CO, FT, SOC1, AP1, LFY, FLC, and TFL1 are regulated [9,14,51]. We collected samples at 0, 1, 3, 5, and 7 d after 50 mg L −1 GV and control (0 mg L −1 GV) treatment. Our results showed the transcript levels of the floral regulatory genes SOC1, LFY, and AP1 were significantly upregulated (2~6 foldchange) compared to the control after GV treatment in Arabidopsis ( Figure 5A-C). GI, CO, FT, FLC, and TFL1 showed only weak upregulation at some points ( Figure S8B-F), suggesting that SOC1, AP1, and LFY are the key flowering regulators induced by GV. In the 35S:WRKY41 and 35S:WRKY53 lines, the related transcript levels of SOC1, LFY, and AP1 were significantly elevated compared to WT plants ( Figure 5D-F), indicating that they are the primary flowering regulators induced by WRKY41 and WRKY53. To further verify whether WRKY41 and WRKY53 have effects on the transcriptional activation of SOC1, LFY, and AP1, a dual-luciferase (Luc)-based reporter assay was conducted in Nicotiana benthamiana. The WRKY41 and WRKY53 proteins acted as the effectors and the 2-kb promoter regions of SOC1, LFY, and AP1 were the reporters. We found that in the presence of WRKY41 or WRKY53, the expression of luciferase driven by the native SOC1 and LFY promoters was greater than that driven by the same promoters carrying mutated W-boxes ( Figures 5G-J and S9B-D), but AP1 was not activated (Figure S10), suggesting that WRKY41 and WRKY53 mainly regulate the expression of SOC1 and LFY. SOC1 and LFY expression levels were also measured in the w41/w53 lines via qPCR. The results showed that SOC1 and LFY are down-regulated in the w41/w53 lines compared to the WT with and without GV treatment, respectively ( Figure 5K,L), again showing regulation of SOC1 and LFY by WRKY41 and WRKY53, respectively. In addition, SOC1 and LFY were detected at low levels in the w41/w53 lines compared to that in WT after GV treatment, indicating that the upregulation of SOC1 and LFY by GV is mediated by WRKY41 and WRKY53. Together, these results indicated that WRKY41 and WRKY53 mediate GV-induced expression of SOC1 and LFY. based reporter assay was conducted in Nicotiana benthamiana. The WRKY41 and WRKY53 proteins acted as the effectors and the 2-kb promoter regions of SOC1, LFY, and AP1 were the reporters. We found that in the presence of WRKY41 or WRKY53, the expression of luciferase driven by the native SOC1 and LFY promoters was greater than that driven by the same promoters carrying mutated W-boxes (Figures 5G-J and S9B-D), but AP1 was not activated ( Figure S10), suggesting that WRKY41 and WRKY53 mainly regulate the expression of SOC1 and LFY. SOC1 and LFY expression levels were also measured in the w41/w53 lines via qPCR. The results showed that SOC1 and LFY are down-regulated in the w41/w53 lines compared to the WT with and without GV treatment, respectively ( Figure 5K,L), again showing regulation of SOC1 and LFY by WRKY41 and WRKY53, respectively. In addition, SOC1 and LFY were detected at low levels in the w41/w53 lines compared to that in WT after GV treatment, indicating that the upregulation of SOC1 and LFY by GV is mediated by WRKY41 and WRKY53.
Together, these results indicated that WRKY41 and WRKY53 mediate GV-induced expression of SOC1 and LFY.

WRKY41 and WRKY53 Directly Bind to the Promoters of SOC1 and LFY
The findings that WRKY41 and WRKY53 mediate GV-induced SOC1 and LFY expression prompted us to investigate the relationship between the two transcription factors and the two flowering regulatory genes. We first tested whether WRKY41 and WRKY53 directly regulate SOC1 and LFY transcription by binding to the promoter regions. Analysis of the 2-kb promoter regions of SOC1 and LFY showed that they contained one and six W-box elements, respectively ( Figure 6A). We then performed an electrophoresis mobility shift assay (EMSA) to confirm in vitro interactions between GST-tagged WRKY41 and WRKY53 proteins and 200-bp probes containing the W-box elements found in the promoters of SOC1 and LFY. A 200-bp probe without the W-box elements was used as a control. It was evident that both the GST-WRKY41 and GST-WRKY53 proteins could strongly bind to the W-box element in the SOC1 promoter ( Figures 6B and S11B), and they were also shown to strongly bind to the W-box elements in the LFY promoter ( Figures 6C and S11C). This indicated that WRKY41 and WRKY53 were able to directly bind to the promoters of SOC1 and LFY through the W-box elements.  We next performed chromatin immunoprecipitation (ChIP)-qPCR analysis using the transgenic 35S:WRKY41-GFP and 35S:WRKY53-GFP lines to determine whether WRKY41 and WRKY53 could directly bind to the SOC1 and LFY promoters in vivo. Consistent with the results in vitro, WRKY41 and WRKY53 were both bound to the W-box element in the SOC1 promoter ( Figures 6D and S11D); WRKY41 was bound to the LFY-1 and LFY-3 W-box element, and WRKY53 was bound to the LFY-1 W-box element in the LFY promoter ( Figures 6E and S11E). These results suggest that WRKY41 and WRKY53 can directly bind to SOC1 and LFY promoters, in vivo.

Mutations in SOC1 and LFY Suppress Early Flowering in 35S:WRKY41 Lines
The results described above suggested that WRKY41 and WRKY53 likely promote flowering by directly activating the transcription of SOC1 and LFY. To determine the genetic relationship between WRKY41/WRKY53 and SOC1/LFY in flowering regulation, we generated SOC1 and LFY knockout lines in both WT and 35S:WRKY41 ( Figure S4C) backgrounds using the CRISPR/Cas9-mediated genome editing system. Sequencing confirmed that homozygous soc1, 35S:WRKY41-soc1, lfy, and 35S:WRKY41-lfy lines were obtained ( Figure 7A,B). The flowering phenotypes of soc1 ( Figure 7C,E,F) and lfy ( Figure 7D-F) were similar to those of the previously published soc1-2 [13] and lfy-1 [52] lines, respectively, and later compared to WT, indicating that the selected editing sites in SOC1 and LFY were effective. Consistent with our findings that SOC1 and LFY were directly downstream of WRKY41, the early flowering phenotype caused by overexpression of WRKY41 was fully repressed in the 35S:WRKY41-soc1 ( Figure 7C,E,F and Table S1) and 35S:WRKY41lfy lines ( Figure 7D-F and Table S1). These results suggested that the early flowering in 35S:WRKY41 is mainly attributable to the induction of SOC1 and LFY expression. Moreover, the flowering time of soc1 and lfy lines could not be improved after GV treatment ( Figure S12). These results revealed that WRKY41 mediates GV-induced flowering in a SOC1/LFY-dependent manner. and LFY were effective. Consistent with our findings that SOC1 and LFY were directly downstream of WRKY41, the early flowering phenotype caused by overexpression of WRKY41 was fully repressed in the 35S:WRKY41-soc1 ( Figure 7C,E,F and Table S1) and 35S:WRKY41-lfy lines ( Figure 7D-F and Table S1). These results suggested that the early flowering in 35S:WRKY41 is mainly attributable to the induction of SOC1 and LFY expression. Moreover, the flowering time of soc1 and lfy lines could not be improved after GV treatment ( Figure S12). These results revealed that WRKY41 mediates GVinduced flowering in a SOC1/LFY-dependent manner.

Discussion
Plant growth and development are affected by a variety of biotic and abiotic stresses. To ensure reproductive success and complete seed development under favorable natural conditions, controlled regulation of flowering has been considered an essential measure that is used in agriculture [7]. The promotion of plant flowering has been shown to protect plants against harsh environmental conditions, such as pathogen attacks, drought, heat, and frost, which endanger seed production and harvesting [53,54]. Recent studies have confirmed that plant flowering is affected by PGRs, for Values are expressed as means ± SD (n = 15). Similar results were obtained from three biological repeats. Different letters above the bars indicate statistically significant differences (adjusted p < 0.05, one-way ANOVA). Bar = 1 cm.

Discussion
Plant growth and development are affected by a variety of biotic and abiotic stresses. To ensure reproductive success and complete seed development under favorable natural conditions, controlled regulation of flowering has been considered an essential measure that is used in agriculture [7]. The promotion of plant flowering has been shown to protect plants against harsh environmental conditions, such as pathogen attacks, drought, heat, and frost, which endanger seed production and harvesting [53,54]. Recent studies have confirmed that plant flowering is affected by PGRs, for example, exogenous application of cytokinin (6-BA) promotes flowering in Arabidopsis [5]. Gibberellic (GA) played an important role in accelerating flowering [11,13,55]. In the present study, we confirmed the function of a novel PGR, guvermectin (GV), in inducing early flowering (Figures 1 and S1A,B). Although GV is a nucleoside analog like cytokinin, three cytokinin receptor mutants ahk2/3, ahk2/4, and ahk3/4 all showed a response to GV and flowering early after GV treatment ( Figure S1C,D), suggesting GV is different from cytokinin and acts independent cytokinin signaling or downstream of cytokinin receptors in regulating flowering. Our findings suggested that GV could act as an important new exogenous factor to regulate plant flowering. Biochemical and genetic studies showed that WRKY41 and WRKY53 act as positive regulators of flowering and were induced by GV, and they were shown to comediate GV-accelerated flowering by directly activating the transcription of the flowering regulation genes SOC1 and LFY in Arabidopsis.
Over the past decade, substantial progress has been achieved in defining the roles of WRKY TFs in various stress responses and plant development [19,23,24]. Accumulating data have shown that many WRKY genes are induced by and involved in PGRs-regulated plant growth and development. For example, AtWRKY46, AtWRKY54, and AtWRKY70 are positively involved in BR-regulated growth in plants [46]. Auxin antagonizes leaf senescence through AtWRKY57 [56]. Moreover, extensive studies have shown that WRKY genes, acting as positive or negative regulators, regulate flowering [4,19]. AtWRKY75, a positive regulator, accelerated flowering in Arabidopsis [4]. AtWRKY12 and AtWRKY13 modulate flowering time in opposite directions by directly targeting FRUITFUL (FUL) [19].
Here, an analysis of transcriptome data after GV treatment revealed that multiple WRKY genes were induced (Table S2), and qPCR confirmed that those WRKY genes were induced at different levels in response to GV treatment ( Figure S3B-K). As the highly upregulated WRKY gene in response to GV treatment, WRKY41 (Figure 2A) was selected for further study. A member of WRKY group III, WRKY41 has been reported to play an important role in the regulation of plant growth and development, such as regulating seed dormancy [49]. Studies have shown WRKY41 is expressed in different tissues of the plant, importantly, WRKY41 is expressed in floral tissue [49], suggesting that it may play a role in flower development, but this needs to be confirmed. Here, we found that WRKY41 did indeed function as a new flowering regulator. 35S:WRKY41 plants showed an early flowering phenotype compared to the WT ( Figure 2C-E), providing evidence that WRKY41 positively regulated flowering. However, the flowering time of wrky41 was consistent with that of WT ( Figure 2F-H), implying the existence of a functionally redundant WRKY gene. WRKY TFs have been shown to function redundantly in regulating flowering; for example, AtWRKY71 is functionally redundant with two closely related homologs, AtWRKY8 and AtWRKY28, in regulating flowering [21]. A constructed phylogenetic tree indicated that WRKY53 was the closest homolog to WRKY41 ( Figure S6), interestingly, WRKY53 was also upregulated after GV treatment in Arabidopsis ( Figure 3A). WRKY53 belongs to WRKY group III and has roles in regulating leaf senescence [57], plant disease resistance [58], and plant architecture and seed size in rice [59]. Moreover, WRKY53 is involved in flowering through an unclear mechanism [21]. Our results showed that 35S:WRKY53 lines exhibited an early flowering phenotype compared to WT lines ( Figure 3C-E), indicating that WRKY53 also plays a role in flowering regulation. Notably, w41/w53 double knockouts showed delayed flowering compared to WT ( Figure 4C-E), revealing that WRKY41 and WRKY53 have redundant functions in flowering regulation. Previous studies have shown that WRKY TFs mediate PGRs-induced flowering. For example, the absence of AtWRKY75 leads to a delay in GA-mediated flowering time [4]; wrky12 mutants show less sensitivity to the GA-induced flowering response, but wrky13 mutants are more sensitive to that [19]. Consistently, we found that wrky41 and wrky53 mutants had delays in GV-accelerated flowering time compared to WT plants ( Figures 2F-H and 3F-H), indicating that WRKY41 and WRKY53 both play roles in the process of GV-accelerated flowering. Notably, the undifferentiated late-flowering phenotype of w41/w53 lines with and without GV treatment ( Figure 4C-E) provided evidence that WRKY41 and WRKY53 jointly mediate GV-accelerated flowering. We cannot exclude the possibility that other GV-responsive WRKY genes affect flowering; however, the loss of both WRKY41 and WRKY53 function suppressed GV-accelerated flowering ( Figure 5), indicating that other WRKY genes may not be involved in this process. For example, we found the overexpression WRKY61, a WRKY gene responds highly to GV and showed no effect on flowering time ( Figure S5), but the reduction of TCV viral accumulation in overexpression WRKY61 lines indicates that WRKY61 mainly responds to stress [60]. This implies that GV would be able to induce a plant defense response-a promising topic for future study.
It has been reported that major flowering regulatory genes can be induced by a variety of factors that are dependent or not dependent on the flowering pathway in Arabidopsis [20,61,62]. It has also been confirmed that WRKY proteins regulate the expression of different floral integrators or floral meristem identity genes. For example, WRKY71 directly activates FT and LFY [21]; WRKY12 and WRKY13 directly regulate FUL [19]. WRKY75 directly activates FT [4]. We found that SOC1, LFY, and AP1 were all significantly upregulated in GV-treated WT, 35S:WRKY41, and 35S:WRKY53 plants ( Figure 5A-F). Furthermore, a dual-luciferase reporter assay showed direct regulation of SOC1 and LFY expression by WRKY41 and WRKY53 Figures 5G-J and S9B-D). SOC1 has a central role in the transition to flowering [14,62]. Studies have shown that SOC1 is involved in regulating flowering time, floral patterning, and floral meristem (FM) determinacy. Furthermore, SOC1 is not only induced by exogenous factors such as GA [63] and cytokinin [5] but can also be directly regulated by endogenous genes such as FT [64], miR172 [65], and NUCLEAR FACTOR Y [13] through direct binding to its promoter. Although it has been demonstrated that SOC1 can be regulated by a variety of factors, there was no prior evidence of a WRKY gene directly regulating SOC1. Interestingly, our EMSA and Chip-qPCR experiments demonstrated, for the first time, that WRKY41 and WRKY53 can directly bind to the SOC1 promoter ( Figures 6B,D and S11B,D). These results add WRKY genes to the list of known genes that directly regulate SOC1 expression. LFY is a target gene of SOC1 and is reportedly involved in regulating flowering time and FM determinacy [9]. As expected, we confirmed that WRKY41 and WRKY53 also directly bind to the LFY promoter ( Figures 6C,E and S11C,E). These results suggest that SOC1 and LFY are target genes of WRKY41 and WRKY53 and that high AP1 expression may be caused by LFY. In genetic phenotype, studies have shown that the loss-functions of SOC1 or LFY, such as soc1-2 lines and lfy-2 lines, delays flowering, and that SOC1 or LFY overexpression leads to early flowering in Arabidopsis [21,66]. In our present study, the loss function of WRKY41 or WRKY53 both weakened the GVinduced early flowering ( Figures 2F and 3F), meanwhile, the early flowering phenotypes of 35S:WRKY41 lines were reversed in the soc1 or lfy background ( Figure 7C-F), implying that WRKY41 and WRKY53 accelerated flowering in a manner dependent on SOC1/LFY. Together, these observations provide supporting evidence that WRKY41 and WRKY53 co-mediate GV-accelerated flowering by directly activating the transcription of SOC1 and LFY in Arabidopsis.

Plant Materials and Growth Conditions
All Arabidopsis plants are in the Col background. Plants were grown under cool white fluorescent lights (80-100 µmol m −2 s −1 ) at 22 • C and 60% relative humidity in incubators (LEDIAN, Ningbo, China) [44]. The long-day (LD) conditions consisted of a 16 h light/8 h dark and short-day (SD) conditions consisted of an 8 h light/16 h dark photoperiod. Col-0 Arabidopsis was used as the wild type (WT). The T-DNA insertion knockout mutants wrky41 (Salk_068648) and wrky53 (Salk_034157) [67] were provided by the AraShare Arabidopsis Stock Centre (Fuzhou, China). Overexpression lines 35S:WRKY41-GFP and 35S:WRKY53-GFP were generated by cloning the full-length WRKY41 and WRKY53 coding sequence (CDS) into the pCHF3 vector, which contains a 35S promoter and a GFP-tag used for screening positive overexpressing plants by western blot and performing ChIP-qPCR assays with anti-GFP antibodies [68,69]. The following mutants were obtained by using CRISPR/cas9-mediated knockout technology [70,71], the w41/w53-1 and w41/w53-2 double mutants were generated by knocking out WRKY53 in wrky41 lines and WRKY41 in wrky53 lines, respectively. The 35S:WRKY41-soc1 and 35S:WRKY41-lfy lines were generated by knocking out SOC1 and LFY, respectively, in 35S:WRKY41 lines. The soc1 and lfy lines were generated by knocking out SOC1 and LFY in the WT.

Gene Expression Analysis
Two-week-old Arabidopsis thaliana leaves were sprayed with 50 mg L −1 guvermectin (GV) once, and the whole plant was ground with liquid nitrogen at 0, 1, 3, 5, and 7 d after GV treatment, then total RNA was extracted using TRIzol reagent [74]. Meanwhile, the plants sprayed with 0 mg L −1 GV were used as a control at each point. For qRT-PCR, 1 µg total RNA per sample was treated with the PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, Takara, San Jose, CA, USA). qRT-PCR was performed with SYBR Master Mix (Q711-03, Vazyme, Nanjing, China) on the Bio-Rad iQ5 optical system software (Bio-Rad, Hercules, CA, USA). UBQ5 was used as the internal control gene for expression level normalization. The transcript level of each gene was calculated using the double ∆Ct method [34]. Data analysis was conducted in GraphPad Prism 8. The primers were summarized in Table S3.

Transcriptome Analysis
RNA sequencing (RNA-seq) was performed according to Xie et al. [75], with minor modifications. Two-week-old Arabidopsis thaliana grown under LD conditions were treated by spraying with 50 mg L −1 GV once and the plants sprayed with 0 mg L −1 GV were used as a control. The whole plant, four days after GV treatment, was collected for RNA extraction and RNA-seq. Sequencing was performed on an Illumina NovaSeq 6000 (Illumine, Austin, TX, USA). Three independent biological replicates were sequenced and analyzed.

Electrophoretic Mobility Shift Assay (EMSA)
To investigate the interaction between the transcription factors WRKY41 and WRKY53 and the promoters of SOC1 and LFY, The EMSA was performed as described previously [76,77] The CDSs of WRKY41 and WRKY53 were inserted into a vector containing the GST tag. GST-WRKY41 and GST-WRKY53 proteins were then expressed and purified using GST Protein Purification System (GE Healthcare, Chicago, IL, USA). The NDA Probes 200 bp (10 ng) generated based on the promoter sequences of SOC1 and LFY and various concentrations of GST-WRKY41 and GST-WRKY53 were incubated in 20 µL binding buffer (20 mmol/L Tris base, 5% (v/v) glycerol, 2 mmol/L dithiothreitol, 5 mmol/L MgCl 2 , and 0.5 µg BSA) at 25 • C for 25 min. The reaction mixtures were analyzed with 4% nondenaturing polyacrylamide gel after electrophoretic at 4 • C. The DNA-protein complexes were observed after incubation with the addition of SYBR GOLD chemiluminescent dyes for 1 h and photographed under ultraviolet transillumination. GST was used as the negative control. The primers were summarized in Table S3.

ChIP-qPCR Assay
Chromatin immunoprecipitation (ChIP) was performed as previously described [78,79], with minor modifications. Approximately 4 g of four-week-old plant tissue per sample was used for ChIP-qPCR analysis. The plant lines analyzed were 35S:WRKY41-GFP, 35S:WRKY53-GFP, and WT. The protein-DNA complexes of 35S:WRKY41-GFP, 35S:WRKY53-GFP, and WT lines were incubated with GFP-Trap Agarose Beads (ChromoTek). The enrichment of DNA fragments was determined by qPCR. Three independent biological replicates were performed. The primers were listed in Table S3.

Dual-Luciferase Assay
DNA segments (2 kb in length) of the SOC1 and LFY promoters were cloned and inserted into pGreenII 0800-Luc to activate the Luc reporter gene. This plasmid carries a Renilla luciferase gene (REN) as the internal control. The CDS of WRKY41 and WRKY53 were also inserted into pGreenII-62-SK to serve as effectors. Each vector was transformed into A. tumefaciens GV3101 (pSoup), and cells were transfected into the N. benthamiana after mixing in a ratio of 1:9. The activity of Luc and Ren were detected with a multifunctional microplate reader at 48 h after transfection, respectively. The ratio of Luc:Ren was calculated as previously described [67,72,80] with minor modifications. The primers were listed in Table S3.

The Phylogenetic Construction
The phylogenetic construction is according to WU et al. [50]. The Arabidopsis WRKY genes were obtained in NCBI GenBank (https://www.ncbi.nlm.nih.gov/gene/, accessed on 4 May 2023), and then the phylogenetic tree was constructed using Software MEGA6.0.6.

Conclusions
In summary, our results confirmed the biological activity of guvermectin (GV) in accelerating flowering. The expression of transcription factors WRKY41 and WRKY53 were significantly induced by GV, suggesting they may play a role. The phylogenetic tree results indicated that WRKY53 is the closest homolog to WRKY41. Overexpression of WRKY41 or WRKY53 lines showed an early flowering phenotype and the double knockout mutants w41/w53 lines showed a late-flowering phenotype in Arabidopsis, indicating WRKY41 and WRKY53 play an important role in regulating flowering. Meanwhile, after GV treatment, wrky41 or wrky53 lines showed a weakened early flowering than WT and w41/w53 lines showed a late-flowering phenotype, which provided evidence that WRKY41 and WRKY53 jointly mediate GV-accelerated flowering. Gene expression analysis showed GV induced the expression of SOC1 and LFY through WRKY41 and WRKY53. Furthermore, we confirmed that SOC1 and LFY are both direct targets of WRKY41 and WRKY53. Together, these results revealed that WRKY41 and WRKY53 co-mediate GV-accelerated flowering by directly activating the transcription of SOC1 and LFY in Arabidopsis (Figure 8).